Assay Procedure for Lipase

Principle

The appearance of quinoneimine dye is measured at 545 nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of glycerol (half a micromole of quinoneimine dye) per minute under the conditions described below.

Method

Reagents

Procedure

(1st step)

1. Pipette 2.0 mL of olive oil emulsion (A) into a test tube and equilibrate at 37 ℃ for about 5 minutes.

2. Add 0.2 mL of the enzyme solution* and mix.
3. After exactly 15 minutes at 37 ℃, add 2.0 mL of TCA solution (E) to stop the reaction and remove the precipitate by filtration through filter paper (Toyo-Roshi No.131 or Whatman No.42).
   

(2nd step)

4. Pipette 0.05 mL of the filtrate thus obtained into a test tube.
5. Add 3.0 mL of color developing reagent (G) and incubate at 37 ℃ for 15 minutes.
   
6. Measure the optical density at 545 nm against water (OD test).
   

At the same time, prepare the blank by first mixing 2.0 mL of the olive oil emulsion (A) after 15 min incubation at 37 ℃ with 2.0 mL of TCA solution, followed by the addition of the enzyme solution (first step). By using the filtrate obtained from the mixture, carry out the 2nd step using the same procedure as the test and measure the optical density at 545 nm (OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (H) and dilute to 0.4－1.2 U/mL with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula：

Weight activity (U/mg)＝(U/mL)×1/C

This procedure is for informational purposes.